A novel Pik allele confers extended resistance to rice blast.

In the ongoing arms race between rice and Magnaporthe oryzae, the pathogen employs effectors to evade the immune response, while the host develops resistance genes to recognise these effectors and confer resistance. In this study, we identified a novel Pik allele, Pik-W25, from wild rice WR25 through bulked-segregant analysis, creating the Pik-W25 NIL (Near-isogenic Lines) named G9. Pik-W25 conferred resistance to isolates expressing AvrPik-C/D/E alleles. CRISPR-Cas9 editing was used to generate transgenic lines with a loss of function in Pik-W25-1 and Pik-W25-2, resulting in loss of resistance in G9 to isolates expressing the three alleles, confirming that Pik-W25-induced immunity required both Pik-W25-1 and Pik-W25-2. Yeast two-hybrid (Y2H) and split luciferase complementation assays showed interactions between Pik-W25-1 and the three alleles, while Pik-W25-2 could not interact with AvrPik-C, -D, and -E alleles with Y2H assay, indicating Pik-W25-1 acts as an adaptor and Pik-W25-2 transduces the signal to trigger resistance. The Pik-W25 NIL exhibited enhanced field resistance to leaf and panicle blast without significant changes in morphology or development compared to the parent variety CO39, suggesting its potential for resistance breeding. These findings advance our knowledge of rice blast resistance mechanisms and offer valuable resources for effective and sustainable control strategies.

The Piks allele of the NLR immune receptor Pik breaks the recognition of AvrPik effectors of rice blast fungus.

Arms race co-evolution of plant-pathogen interactions evolved sophisticated recognition mechanisms between host immune receptors and pathogen effectors. Different allelic haplotypes of an immune receptor in the host mount distinct recognition against sequence or non-sequence related effectors in pathogens. We report the molecular characterization of the Piks allele of the rice immune receptor Pik against rice blast pathogen, which requires two head-to-head arrayed nucleotide-binding sites and leucine-rich repeat proteins. Like other Pik alleles, both Piks-1 and Piks-2 are necessary and sufficient for mediating resistance. However, unlike other Pik alleles, Piks does not recognize any known AvrPik variants of Magnaporthe oryzae. Sequence analysis of the genome of an avirulent isolate V86010 further revealed that its cognate avirulence (Avr) gene most likely has no significant sequence similarity to known AvrPik variants. Piks-1 and Pikm-1 have only two amino acid differences within the integrated heavy metal-associated (HMA) domain. Pikm-HMA interacts with AvrPik-A, -D, and -E in vitro and in vivo, whereas Piks-HMA does not bind any AvrPik variants. Characterization of two amino acid residues differing Piks-1 from Pikm-1 reveal that Piks-E229Q derived from the exchange of Glu229 to Gln229 in Piks-1 gains recognition specificity against AvrPik-D but not AvrPik-A or -E, indicating that Piks-E229Q partially restores the Pikm spectrum. By contrast, Piks-A261V derived from the exchange of Ala261 to Val261 in Piks-1 retains Piks recognition specificity. We conclude that Glu229 in Piks-1 is critical for Piks breaking the canonical Pik/AvrPik recognition pattern. Intriguingly, binding activity and ectopic cell death induction is maintained between Piks-A261V and AvrPik-D, implying that positive outcomes from ectopic assays might be insufficient to deduce its immune activity against the relevant effectors in rice and rice blast interaction.

Characterization and proposed spontaneous deletion mechanism of AVR-Pik locus in Pyricularia oryzae.

In phytopathogenic fungi, a mutation in the avirulence gene can lead to the breakdown of resistance in the host plant. The nucleotide sequences of the AVR-Pik locus in the strain Ina168 and its spontaneous mutant Ina168m95-5 of Pyricularia oryzae were determined. An AVR-Pik spontaneous deletion mechanism of Ina168m95-5, including multiple homologous recombination events involving repetitive transposable elements, is proposed.

Novel haplotypes and networks of AVR-Pik alleles in Magnaporthe oryzae.

BACKGROUND: Rice blast disease is one of the most destructive fungal disease of rice worldwide. The avirulence (AVR) genes of Magnaporthe oryzae are recognized by the cognate resistance (R) genes of rice and trigger race-specific resistance. The variation in AVR is one of the major drivers of new races. Detecting the variation in the AVR gene in isolates from a population of Magnaporthe oryzae collected from rice production fields will aid in evaluating the effectiveness of R genes in rice production areas. The Pik gene contains 5 R alleles (Pik, Pikh, Pikp, Pikm and Piks) corresponding to the AVR alleles (AVR-Pik/kh/kp/km/ks) of M. oryzae. The Pik gene specifically recognizes and prevents infections by isolates of M. oryzae that contain AVR-Pik. The molecular variation in AVR-Pik alleles of M. oryzae and Pik alleles of rice remains unclear. RESULTS: We studied the possible evolutionary pathways of AVR-Pik alleles by analyzing their DNA sequence variation and assaying their avirulence to the cognate Pik alleles of resistance genes under field conditions in China. The results of PCR products from genomic DNA showed that 278 of the 366 isolates of M. oryzae collected from Yunnan Province, China, carried AVR-Pik alleles. Among the isolates from six regions of Yunnan, 66.7-90.3% carried AVR-Pik alleles. Moreover, 10 AVR-Pik haplotypes encoding five novel AVR-Pik variants were identified among 201 isolates. The AVR-Pik alleles evolved to virulent from avirulent forms via stepwise base substitution. These findings demonstrate that AVR-Pik alleles are under positive selection and that mutations are responsible for defeating race-specific resistant Pik alleles in nature. CONCLUSIONS: We demonstrated the polymorphism and distribution of AVR-Pik alleles in Yunnan Province, China. By pathogenicity assays used to detect the function of the different haplotypes of AVR-Pik, for the first time, we showed the avoidance and stepwise evolution of AVR-Pik alleles in rice production areas of Yunnan. The functional AVR-Pik possesses diversified sequence structures and is under positive selection in nature.

Magnaporthe oryzae Effector AvrPik-D Targets Rice Rubisco Small Subunit OsRBCS4 to Suppress Immunity.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae (M. oryzae), is a highly destructive disease that significantly impacts rice yield and quality. During the infection, M. oryzae secretes effector proteins to subvert the host immune response. However, the interaction between the effector protein AvrPik-D and its target proteins in rice, and the mechanism by which AvrPik-D exacerbates disease severity to facilitate infection, remains poorly understood. In this study, we found that the M. oryzae effector AvrPik-D interacts with the Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) small subunit OsRBCS4. The overexpression of the OsRBCS4 gene in transgenic rice not only enhances resistance to M. oryzae but also induces more reactive oxygen species following chitin treatment. OsRBCS4 localizes to chloroplasts and co-localizes with AvrPik-D within these organelles. AvrPik-D suppresses the transcriptional expression of OsRBCS4 and inhibits Rubisco activity in rice. In conclusion, our results demonstrate that the M. oryzae effector AvrPik-D targets the Rubisco small subunit OsRBCS4 and inhibits its carboxylase and oxygenase activity, thereby suppressing rice innate immunity to facilitate infection. This provides a novel mechanism for the M. oryzae effector to subvert the host immunity to promote infection.